Uphill energy transfer mechanism for photosynthesis in an Antarctic alga

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Å resolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC’s unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.


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March 2021

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Replication
The initial model was built using Map_to_Model in PHENIX software (doi: 10.1038/s41592-018-0173-1, doi: 10.1107/S2059798319011471). The model was manually corrected by Coot (doi: 10.1107/S0907444904019158), followed by Real-space Refinement in PHENIX (doi: 10.1107/ S2059798318006551). The model was refined by multiple cycles of manual modifications in Coot and Real-space Refinement in PHENIX. NCS restraints were used for the automatic refinement. The refined model was validated using MolProbity in PHENIX (doi: 10.1002/pro.3330). FSC between the map and the model were calculated by PHENIX. UCSF Chimera and PyMOL (Schrödinger, New York, NY, USA) were used for visualization. The fitting analysis of absorbance spectrum (Fig.1d) with Gaussian functions was performed by Magic plot 2.7.2 (Magicplot Systems, St. Petersburg, Russia). The kinetic analysis of excitation energy transfer in Fig.8 and Supplementary Fig.6 was conducted using a software igor pro ver. 6 (WaveMetrics, Inc. Portland, USA).The signal peptides and the transmembrane helices of Cr_Lhca2, Bc_LhcaJ and Ds_Lhca5 were predicted from the 3D structures registered in the PDB (Cr_Lhca2; 6JO5, 6IJO, Bc_LhcaJ; 6IGZ, Ds_Lhca5; 6SL5) and from the results of secondary structural prediction using Jpred 4 (doi: 10.1093/nar/gkv332) and TargetP-2.0 (doi: 10.26508/lsa.201900429). Evolutionary analyses of LHCs in Fig. 3 were conducted with the neighbor-joining method in MEGA7 (10.1093/molbev/msw054). Our study did not deal with "sex and gender".

The Cryo-EM map of Pc-frLHC is deposited in the Electron
Our study is not including human research.
Any recruitment was not conducted in our research.
Our study was not needed ethics oversight because green algae are not the target of that.
We did not perform statistical method to determine the sample size. Sample size of the experiments was set as small as possible because the Prasiola crispa sample harvested from Antarctica was limited. The colony size for the measurement of transmittance spectra in Supplementary  Fig. 1b was determined under consideration of the irradiation area and the sensor size. The amount of protein sample used for the biochemical experiments (Figs.1, 8, Supplementary Figs. 1c-e, 2, 3, 5, 6) was modified based on the previous experience to obtain suitable signals.
For single particle analysis, sample size was determined by available machine time of the cryo-EM for data collection. 1,555 micrographs were acquired and the number of particles for Class2D, Class3D and Refine3D were 696,095, 654,477 and 99,510, respectively.
In the process of 2D and 3D classification of single particle analysis, 86% of the total particles were eliminated by the algorithm on RELION3.
Supplementary Fig.1: The transmitted light of samples were determined by a spectrometer 6 times under each condition and averaged.